This invention generally relates to encapsulation of core materials, including viable cells, within an intracapsular volume defined by a semipermeable membrane. More particularly, the invention relates to a process for producing large quantities of capsules having uniform membranes with improved porosity control adapted to promote growth of cells within the capsules.
U.S. Pat. No. 4,352,883, issued Oct. 5, 1982, upon application of Dr. Franklin Lim, discloses a basic procedure for encapsulating core materials, including viable cells, within capsules having semipermeable membranes. Viable cells encapsulated with the Lim procedure are capable of on-going metabolism, including mitosis, and secrete materials they would normally secrete in their unencapsulated form. Capsules made with the Lim technique may be engineered to have membranes which are permeable to molecules below a particular molecular weight but substantially impermeable to higher molecular weight molecules and to cells. The pores of the membranes are believed to comprise tortuous paths defined by the interstices of the membrane structure. Passage of molecules above a particular molecular weight is hindered by these tortuous path pores, and. above a certain higher molecular weight and corresponding effective molecular dimension, the hindrance is sufficiently great that the membrane is substantially impermeable to these molecules.
Porosity control is an important factor in a number of important uses of such microcapsules. The microcapsule membrane can be used for differential screening, that is, to separate molecules on a molecular weight basis. For example, U.S. Pat. No. 4,409,331, issued Oct. 11, 1983, discloses a method wherein substances secreted by cells within the capsule may traverse the membrane while other, higher molecular weight materials are confined within the capsules. Such capsules can simplify greatly collection of a substance of interest. Copending application Ser. No. 485,472, filed Apr. 15, 1983, now U.S. Pat. No. 4,582,799, discloses a process wherein cells producing a substance of interest which is not secreted may be lysed within the capsule membrane. Low molecular weight substances of interest can diffuse across the membrane into the extracapsular medium while cell debris and high molecular weight substances and contaminants, e.g., pyrogens, are trapped within the intracapsular volume.
The selective screening properties of the capsule membrane also allow the capsules to be used for cross-strain in vivo growth of hybridomas, as disclosed in copending application Ser. No. 579,460, filed Feb. 13, 1984, now abandoned. The capsule membrane permits cross-strain hybridomas to be grown within a body cavity of an animal whose immune system would normally attack the hybridomas. Astute engineering of membrane permeability properties allows high specificity collection of the secreted substance.
Effective membrane permeability control also permits the use of implanted capsules containing cells which secrete an antigen as an immunizing agent. The screening properties of the membrane produce relatively pure antigen as the immunizing agent without the need of a tedious antibody purification procedure and can lead to stimulation of specific antibody production.
Capsules with such membranes can also be used as part of a cell screening procedure. Extracapsular medium is tested for a substance secreted through the membrane. Contaminants having a molecular weight greater than the substance are kept within the capsule thereby reducing false positive results.
Application Ser. No. 579,460 No. DBH-455) was filed on even date herewith. This disclosure and the disclosure of U.S. Pat. No. 4,352,883 are incorporated herein by reference.
A preferred embodiment of the Lim encapsulation technique involves the formation of shape-retaining gelled masses which contain the material to be encapsulated, followed by deposition of a membrane on the surface of the gelled masses. The membrane is formed as relatively high molecular weight materials contact the gel masses and form ionic cross-links with the gel. Lim discloses that lower molecular weight cross-linking polymers permeate further into the structure of the gelled masses and result in a reduction of pore size. Lim also discloses that the duration of membrane formation affects pore size. Given a pair of reactants, the longer the cross-linking polymer solution is exposed to the gelled mass, the thicker and less permeable the membrane.
While the techniques for porosity control and membrane formation disclosed in the Lim patent can form acceptable membranes, many of the foregoing applications of the capsule technology could be improved if membranes having improved porosity control and better uniformity could be produced. The Lim porosity control techniques do not allow fine tuning of the membrane porosity, but rather set rough differential filtering limits.
In addition to improved porosity, for commercial purposes it is also important to be able to consistently produce microcapsules in large numbers having defect-free membranes. In this regard, membranes formed by the Lim techniques occasionally have protruding portions of cells or have cells anchored on the capsules. The Lim techniques also may produce capsules containing voids which allow cells, the substance of interest, or unwanted contaminants to escape from the capsule. If some small fraction of the microcapsules made with a specific purpose in mind have membrane voids, many of the objectives and advantages of the processes would be frustrated. Accordingly, modifications of the encapsulation processes which promote membrane uniformity and avoid random membrane defects are advantageous to commercial practice of many of the foregoing processes.
Accordingly, it is object of this invention to improve porosity control of microcapsule membranes. Another object is to promote more uniform membrane formation. A further object is to develop a process allowing formation of membranes which optimize cellular growth and secretion of substances produced by the cells. Still another object of the invention is to provide a process for producing permeable capsule membranes having more precise permeability limits and for reproducibly engineering such limits. Other objects and features of the invention will be apparent from the following.